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UDP glucuronate decarboxylase activity was comparable in encapsulated and non-encapsulated strains of Cryptococcus neoformans, required NAD (K(a) = 0.2 mM), and was inhibited by NADH (K(i) = 0.1 mM) and UDP xylose.
Investigations into mechanisms of binding of encapsulated and acapsulate strains of Cryptococcus neoformans by human neutrophils were performed, using a monolayer assay. The two strains bound to neutrophils by different mechanisms although both had an absolute requirement for opsonization with complement components in normal human serum for binding to occur. Neutrophil binding of encapsulated yeasts required conformational changes in actin yet did not appear to lead to phagocytosis of the organism. A maximum of 12 acapsulate cells bound per neutrophil compared with only four encapsulated yeasts. Cytochalasin D treatment reduced the maximum numbers able to bind per neutrophil by 50%. The encapsulated cryptococci appeared to compete with each other for binding to neutrophils whereas the acapsulate yeast cells bound to neutrophils in an approximately Poisson distribution, suggesting independent binding. Binding of acapsulate cryptococci did not require actin filaments and appeared to trigger phagocytosis. Thus, the capsule of C. neoformans appeared to inhibit binding and internalization by neutrophils.
Natural killer (NK) cells bind to and inhibit the growth of the fungal target Cryptococcus neoformans. Since C. neoformans is structurally and chemically distinct from the standard tumor cell target used in the model of NK cell-mediated cytotoxicity, this study was designed to investigate the NK cell response after binding to cryptococci. Transmission electron micrographs and three-dimensional reconstructions of NK cell-cryptococci conjugates demonstrated focusing of the NK cell centrioles and Golgi apparatus toward the cryptococcal attachment site. NK cell cytoskeletal changes after cryptococcal binding were confirmed by immunofluorescence studies in which NK cells were allowed to bind to cryptococci in Mg2(+)-containing, Ca2(+)-free medium. One hour after the addition of Ca2+ to the preformed conjugates, the bound NK cells demonstrated a significant increase in the percentage of microtubule organizing centers focused toward the cryptococcal binding site. Colchicine, a drug that inhibits microtubule assembly, did not affect NK cell-cryptococci binding but abrogated NK cell-mediated cryptococcal growth inhibition, indicating that microtubule assembly, an important prerequisite for the secretory process, is not required for NK cell-cryptococci binding but is essential for inhibition of cryptococcal growth. In addition, the Ca2+ channel-blocking reagents, lidocaine and verapamil, did not affect NK cell-cryptococci binding but blocked the NK cell-mediated anticryptococcal activity, suggesting that a Ca2+ flux is essential for inhibition of cryptococcal growth. Considered together, these data indicate that NK cells respond to binding of a target cell that has a capsule and cell wall, in addition to a cell membrane, in a manner similar to that seen following binding to target cells that are surrounded by only a cell membrane; however, the response of the NK cells to the binding of C. neoformans is slower and possibly less efficient than the response after tumor cell binding.
The GPD gene encoding glyceraldehyde-3-phosphate dehydrogenase was isolated from Cryptococcus neoformans, a heterobasidiomycetous yeast that is pathogenic to humans. The gene contains 11 introns, differing from the conserved intron positions found in the GPD genes of Basidiomycetes. The predicted amino-acid sequence of this gene is extremely similar to that reported from GPD proteins of other basidiomycetes. The promoter region of the C. neoformans GPD gene was similar to those of other basidiomycetes. Plasmid constructs containing up to 1600 base pairs upstream of the native GPD open reading frame were used to express either the native URA5 gene in a ura5 mutant or the heterologous hphI gene (a bacterial gene that confers resistance to the aminoglycoside hygromycin) in a wild-type strain of C. neoformans. Transformation frequencies resulting from the plasmid-borne Gpdp::URA5 gene were at levels similar to those of the native URA5, which suggested that all the sequences necessary for proper expression were present. Transformation frequencies using the Gpdp::hphI gene constructs were poor. However, addition of DNA sequences flanking the 3'-end of an native C. neoformans gene significantly improved the transformation frequencies resulting from the expression of the heterologous hphI gene.
A hybridoma (clone CRND-8) that produced agglutinating monoclonal antibody (MAb) against Cryptococcus neoformans serotype D was established by using a soluble capsular polysaccharide-keyhole limpet hemocyanin conjugate for immunization. The isotype was immunoglobulin M(kappa). Specificity was determined by cell slide agglutination and enzyme-linked immunosorbent assay (ELISA). In both tests, the MAb reacted to serotypes D and A-D but not to serotypes A, B, and C. Furthermore, the specificity of the MAb determined by ELISA was the same as that of polyclonal antibody factor serum (PAb factor) 8, which showed high-level reactivity with serotypes D and A-D. These results supported the deduced specificity of the PAb-based antigenic factor 8. A total of 15 isolates of serotypes D and A-D but no serotype A isolates reacted with the MAb in cell slide agglutination tests. CRND-8 MAb can be used in place of PAb factor 8 for serotyping C. neoformans isolates and for the analysis of the antigen 8 epitope.
As an extension of the previously established association between Cryptococcus neoformans var. gattii and Eucalyptus camaldulensis (River red gum) we have now found a similar relationship between C. neoformans var. gattii and the closely related Eucalyptus tereticornis (Forest red gum). The global distribution of E. tereticornis is similar to that of E. camaldulensis.
We have undertaken an analysis of the retrotransposons in the medically important basidiomycetous fungus Cryptococcus neoformans. Using the data generated by a C. neoformans genome sequencing project at the Stanford Genome Technology Center, 15 distinct families of LTR retrotransposons and several families of non-LTR retrotransposons were identified. Members of at least seven families have transposed recently and are probably still active. For several families, only partial elements could be identified and these are quite diverse in sequence, suggesting that they are ancient components of the C. neoformans genome. Most C. neoformans elements are not closely related to previously identified fungal retrotransposons, suggesting that the diversity of fungal retrotransposons has been only sparsely sampled to date. C. neoformans has fewer distinct retrotransposon families than Candida albicans (37 or more), in particular fewer families represented solely by ancient and inactive elements, but it has considerably more families than either Saccharomyces cerevisiae (five) or Schizosaccharomyces pombe (two). The findings suggest that elimination of retrotransposons is faster in C. neoformans than in C. albicans, but perhaps not as rapid as in S. cerevisiae or Sz. pombe. The identification of the retrotransposons of C. neoformans should assist in the molecular characterization of this important pathogen, and also further our understanding of the role played by retroelements in genome evolution.
The effects of a modified natural porcine surfactant (Curosurf) on phagocytosis and killing of Cryptococcus neoformans by alveolar macrophages and on the production of superoxide anions were investigated in vitro. Attachment and ingestion were evaluated separately by a fluorescent quenching technique. The nitroblue tetrazolium reduction test was used as an indirect measurement of superoxide anion production. Killing was assessed by a colony-forming assay. Surfactant induced increased ingestion of C. neoformans, unopsonized as well as opsonized with fresh serum or anticryptococcal polyclonal IgG. Surfactant had, however, no effect on the attachment or killing of unopsonized or opsonized C. neoformans by the alveolar macrophages. In addition, the enhancement of the oxidative metabolism of the macrophages after stimulation with opsonized yeast was impaired, although the killing was not affected. This study indicates that in vitro Curosurf can influence the alveolar macrophage defence against C. neoformans by enhancing its ingestion and by interacting with the superoxide anions release from alveolar macrophages stimulated with fresh serum or anticryptococcal polyclonal IgG opsonized yeast cells.
The fungal pathogens Cryptococcus neoformans and Histoplasma capsulatum produce melanin-like pigments in the presence of L-dopa in vitro and during mammalian infection. We investigated whether melanization affected the susceptibilities of the fungi to amphotericin B, caspofungin, fluconazole, itraconazole, or flucytosine (5FC). Using the standard macrodilution MIC protocol (the M27A protocol) of the National Committee for Clinical Laboratory Standards for yeast, we found no difference in the susceptibilities of melanized and nonmelanized C. neoformans and H. capsulatum isolates. Killing assays demonstrated that melanization reduced the susceptibilities of both fungi to amphotericin B and caspofungin. Laccase-deficient C. neoformans cells grown with L-dopa were significantly more susceptible than congenic melanin-producing yeast to killing by amphotericin B or caspofungin. Preincubation of amphotericin B or caspofungin with melanins decreased their antifungal activities. Elemental analysis of melanins incubated with amphotericin B or caspofungin revealed an alteration in the C:N ratios of the melanins, which indicated binding of these drugs by the melanins. In contrast, incubation of fluconazole, itraconazole, or 5FC with melanins did not significantly affect the antifungal efficacies of the drugs or the chemical composition of the melanins. The results suggest a potential explanation for the inefficacy of caspofungin against C. neoformans in vivo, despite activity in vitro. Furthermore, the results indicate that fungal melanins protect C. neoformans and H. capsulatum from the activities of amphotericin B and caspofungin and that this protection is not demonstrable by standard broth macrodilution assays.
The study of the clinical isolates of Cryptococcus neoformans from 83 Brazilian patients with disseminated cryptococcosis showed that 75 were C. neoformans var. neoformans and 8 were var. gattii. Twenty-seven isolates were serotyped; all 19 var. neoformans were serotype A and all 8 var. gattii were serotype B. The correlation of the varieties of C. neoformans with the presence or not of hosts predisposing conditions to the mycosis showed that: (1) cryptococcosis caused by gattii variety occurred in 7 (58.3%) of the 12 nonimmunosuppressed patients, and (2) cryptococcosis caused by neoformans variety occurred in 65 (98.5%) of the 66 AIDS patients and in all 5 patients with other immunosuppressive conditions. The comparison of the distribution of the gattii and neoformans varieties between the nonimmunosuppressed and immunosuppressed patients showed a significant statistical difference (p < 0.01).
Infection by Cryptococcus neoformans begins with inhalation of infectious propagules. Fungi reach the lung tissue and interact with epithelial cells in a crucial but poorly understood process. In this study, the interaction of C. neoformans with the human alveolar epithelial cell lineage A549 was investigated, focusing on the relevance of the capsular polysaccharide in this process. The association of encapsulated strains with A549 cells was significantly inhibited by a monoclonal antibody to glucuronoxylomannan (GXM), a major component of the cryptococcal capsule. A purified preparation of GXM produced similar results, suggesting the occurrence of surface receptors for this polysaccharide on the surface of alveolar cells. A549 cells were in fact able to bind soluble GXM, as confirmed by indirect immunofluorescence analysis using the anti-polysaccharide antibody. C. neoformans is internalized after GXM-mediated interaction with A549 cells in a process that culminates with death of host cells. Our results suggest that C. neoformans can use GXM for attachment to alveolar epithelia, allowing the fungus to reach the intracellular environment and damage host cells through still uncharacterized mechanisms.
Cryptococcal antigen was detected in the serum of 3 patients with biopsy-proved Cryptococcus neoformans pneumonitis. It is possible that the specificity of this antigen test will allow the serologic diagnosis of cryptococcal pneumonia and obviate the need for invasive procedures.
The organization of the mitochondrial genomes in two strains belonging in different varieties of Cryptococcus neoformans was analysed. Physical maps of the mtDNA of the IFM5844 (var. neoformans) and IFO410 (var. grubii) strains were constructed by using EcoRI and EcoRV restriction enzymes; functional maps were constructed by hybridization, cloning and sequencing. Most of the genes important in the mitochondrial function (ND1, ND2, ND3, ND4, ND4L, ND5, ND6, ATP6, ATP9, COX1, COX2 and COB) and protein synthesis (SsrRNA and LsrRNA) were localized. We did not find any differences between the strains in the order of these genes. However, they differed significantly in the sizes of the mtDNAs: 32.6 kb for IFM5844, and 24.1 kb for IFO410. This can be attributed to two large regions of the mtDNA. In these regions, differences were found in the numbers of introns in COX1 (no intron in var. grubii, 5 introns in var. neoformans), COB (1 intron in var. grubii, 2 introns in var. neoformans), LsrRNA (no intron in var. grubii, 2 introns in var. neoformans), and ND5 (no intron in var. grubii, 1 intron in var. neoformans) genes. In several introns of the COB and COX1 genes LAGLIDADG motifs were found. Differences were also observed in the nucleotide sequences of some genes and in the sizes and sequences of intergenic regions. The nucleotide sequences of the genes of the IFM and IFO strains were compared with those of the H-99 and JEC 21 strains from the database. Surprisingly high similarities were found between the strains belonging in var. grubii (IFO 410 and H-99) and var. neoformans (IFM 5844 and JEC 21).
Sialic acids from sialoglycoconjugates present at the cell surface of Cryptococcus neoformans yeast forms were analyzed by high-performance thin-layer chromatography, binding of influenza A and C virus strains, enzymatic treatment, and flow cytofluorimetry with fluorescein isothiocyanate-labeled lectins. C. neoformans yeast forms grown in a chemically defined medium contain N-acetylneuraminic acid and its 9-O-acetylated derivative. A density of 3 x 10(6) residues of sialic acid per cell was found in C. neoformans. Sialic acids in cryptococcal cells are glycosidically linked to galactopyranosyl units as inferred from the increased reactivity of neuraminidase-treated yeasts with peanut agglutinin. N-Acetylneuraminic acids are alpha-2,6 and alpha-2,3 linked, as indicated by using virus strains M1/5 and M1/5 HS8, respectively, as agglutination probes. The alpha-2,6 linkage markedly predominated. These findings were essentially confirmed by the interaction of cryptococcal cells with the lectins Sambucus nigra agglutinin and Maackia amurensis agglutinin. We also investigated whether the sialyl residues present in C. neoformans are involved in the fungal interaction with a cationic solid-phase substrate and with mouse resident macrophages. Adhesion of yeast cells to poly-L-lysine was mediated, in part, by sialic acid residues, since the number of adherent cells was markedly reduced after treatment with bacterial neuraminidase. The enzymatic removal of sialic acids also made C. neoformans yeast cells more susceptible to endocytosis by macrophages. The results show that sialic acids are components of the cryptococcal cell surface that contribute to its negative charge and protect yeast forms against phagocytosis.
We report the first case of human cryptococcosis due to Cryptococcus neoformans var. gattii described in our country, which was presented as brain cryptococcoma in an immunocompetent patient. An extensive sampling of the patient's environment was carried out to find the source of infection.
The protective efficacy of antibodies (Abs) to Cryptococcus neoformans glucuronoxylomannan (GXM) is dependent on Ab fine specificity. Two clonally related immunoglobulin M monoclonal Abs (MAbs) (12A1 and 13F1) differ in fine specificity and protective efficacy, presumably due to variable (V)-region sequence differences resulting from somatic mutations. MAb 12A1 is protective and produces annular immunofluorescence (IF) on serotype D C. neoformans, while MAb 13F1 is not protective and produces punctate IF. To determine the Ab molecular determinants responsible for the IF pattern, site-directed mutagenesis of the MAb 12A1 heavy-chain V region (V(H)) was followed by serological and functional studies of the various mutants. Changing two selected amino acids in the 12A1 V(H) binding cavity to the corresponding residues in the 13F1 V(H) altered the IF pattern from annular to punctate, reduced opsonic efficacy, and abolished recognition by an anti-idiotypic Ab. Analysis of the binding of the various mutants to peptide mimetics revealed that different amino acids were responsible for GXM binding and peptide specificity. The results suggest that V-region motifs associated with annular binding and opsonic activity may be predictive of Ab efficacy against C. neoformans. This has important implications for immunotherapy and vaccine design that are reinforced by the finding that GXM and peptide reactivities are determined by different amino acid residues.
A novel modified India ink technique for the diagnosis of Cryptococcus neoformans in cerebrospinal fluid specimens is described. It employs 2% chromium mercury and India ink. This technique allows a clear identification of some external and internal structures of the organism. Three layers from the outer capsule that have previously been discerned only by electron microscopy are distinguished.
The capsule is certainly the most prominent virulence factor in Cryptococcus neoformans: acapsular strains are avirulent, and capsular polysaccharides have a deleterious effect on the immune system. Until very recently, very few genes involved in capsule biosynthesis had been identified - and this despite the existence of a detailed body of work concerning the capsule's composition, structure and their regulation by environmental factors. The tremendous development of experimental tools and techniques suited to the study of C. neoformans biology together with the sequencing of three complete genomes have, over the last three years, enabled the identification of a number of proteins which participate directly in biosynthesis of the capsule or which regulate its size. Even though this knowledge is still preliminary, it gives us a clearer picture of the various events needed for biosynthesis of this fascinating structure.